The overall goal of this project is to investigate the role of metabolic factors in mineralization. Previous studies in our laboratories have demonstrated that a transition from an oxidized state to a reduced state at the calcification front is associated with the onset of mineral deposition in the matrix. In the past year, we have mapped the phosphate pools of growth plate cartilage. The data clearly show that the level of nucleotide phosphorus declined as the tissue matured, while the level of inorganic phosphate increased. In parallel with this study, we examined the levels of phosphorus in isolated cells of the normal and rachitic growth cartilage. At the mineralization front, there was a decrease in intracellular phosphorus pools, and a concomitant increase in extracellular phosphate in both normal and rachitic birds. Experiments to measure the effect of vitamin D deficiency on the redox state of the growth plate showed that the level of both NADH and NAD+ were reduced 5-fold when compared to controls, and the transition from the oxidized to the reduced state seen in controls was abolished. To further explore the mechanism by which changes in redox status control mineralization, we propose to test the following hypotheses: 1) the increase in the NADH/NAD+ ratio results from local hypoxia at the calcification front, 2) this change in the results in increased transcellular calcium and phosphate transport and redox ratio 3) changes in cell energy metabolism modulate matrix vesicle biogenesis. These hypotheses will be tested by carrying out a series of in vitro experiments using cultured cells subjected to changes in metabolic status by pharmacological modulation using uncouplers, ionophores and fluoride. Thus, by imposing changes in cellular environment, we expect to learn how cell redox modulates cell mineral metabolism.